This invention relates to the analysis of analytes having a net charge or their derivatives of analytes having a net charge.
Immunoassays can be used to determine the presence and quantity of a range of analytes including antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, and proteins. Their specificity gives these assays particular utility in clinical chemistry. A variety of media are available for performing such assays including, for example, dry immunoassay elements such as multicomponent slides; microtiter plates; xe2x80x9cdip and readxe2x80x9d test strips; bead and tube tests; and microparticles. The different configurations of the immunoassay kits in which the assay is performed are referred to herein as analytical elements. They can comprise, among many others, a bead onto which an antibody is adhered (xe2x80x9cimmobilizedxe2x80x9d) to serve as a receptor, a cup having a surface for similar use, or a polymer layer within a slide onto which sample and reagent are applied, spread, and reacted.
In these assays, conjugate pairs are formed from either analyte (xe2x80x9cligandxe2x80x9d) and receptor combinations or labeled analyte (xe2x80x9clabeled ligandxe2x80x9d) and receptor combinations. In competitive binding immunoassays, labeled ligand competes with unlabeled ligand for reaction with a fixed amount of a particular receptor. Signal measurements such as light absorbance or reflection density are taken of either the bound or unbound labeled ligand after appropriate treatment with various reagents such as chromophores or flourometrically sensitive materials. Unknown ligand concentration is then determined from the measured signal of the labeled ligand after removing (e.g., by washing away) the other species that are not useful for calculating ligand concentration. Thus, it is important to have a reliable method to separate properly bound and unbound forms of ligands or labeled ligands. Failure to do so can lead to inaccurate or imprecise results.
In actual practice, signal measurements can be obtained from labeled ligand, a labeled derivative or analog of the ligand, or a labeled receptor which binds in a specific manner to a ligand as in the case of a sandwich assay.
In general, ligands that are capable of binding non-specifically to, for example, hydrophobic sites and/or ligands that possess a net charge and are capable, therefore, of binding non-specifically to oppositely charged centers are particularly problematic in analytical systems. In such cases, a step may be required to separate free from bound species. That is, they tend to bind to components of assay elements while their labeled counterparts bind to receptors. This makes separation of bound and unbound species incomplete. The same can be said of the labeled ligand when it is the unlabeled ligand that is sought to be bound to the receptor. Aminoglycosides such as gentamicin and tobramycin are two amine rich analytes that possess a net positive charge under conditions in which one or more of the amine groups is protonatedxe2x80x94generally below about pH 11. These analytes are particularly important and yet immunochemical methods of quantitatively measuring them are vulnerable to the inaccuracy and imprecision described above. It is particularly desirable to improve methods for measuring these analytes and others like them.
U.S. Pat. No. 4,547,460 proposes the use of quarternary ammonium compounds as additives for elements of an immunoassay. The ammonium compounds are used to reduce interference from bilirubin and proteins. Presumably, such interference is reduced as a result of complexing bilirubin and/or proteins with the quarternary ammonium compounds. Of course, this can only occur where the bilirubin or protein have a different charge than the ammonium compound.
U.S. Pat. No. 5,279,940 proposes the use of cationic surfactants as signal enhancers in chemiluminescene-based analytical elements. The surfactant is part of the mix of components that provides the enhanced signal. It is not involved in removing substances whose presence would otherwise generate a signal leading to an inaccurate result.
U.S. Pat. No. 4,153,668 proposes using positively charged polymers in an analytical element to more uniformly disperse a liquid containing a negatively charged analyte (a protein bound or proteinaceous substance). The patent discloses only the use of polymers having a net charge that is opposite that of the analyte.
Immunoassay accuracy and precision can still be improved where the analyte has a net charge.
The invention is an assay element for analyzing a charged analyte. The assay element employs an immobilized receptor and a material having a net charge which is the same as that of the analyte.
In one aspect of the invention the analyte is amine rich and the material is a polymer having a net positive charge and the analysis in which the element is used is a competitive binding immunoassay.
In yet another aspect of the invention, the element is a polymer having a net charge such as poly(acrlyamide-co-N-(3-methacrylamidopropyl)-N,N,N-trimethylammonium chloride.
Immunoassays for analytes having a net charge can be improved by the incorporation of a suitable substance possessing the same net charge into analytical element for determining the presence or amount of the analyte. By the same net charge, it is meant that if an analyte possesses a net positive (negative) charge then the substance possesses a net positive (negative) charge, but they are not required to possess the same quantity of charge. By way of illustration, if an analyte possesses a net charge of +2, the substance must possess a net positive charge, but it is not required to be a net charge of +2. It can be any positive charge. Such an element acts to prevent ligand and/or labeled-ligand from adhering nonspecifically. This enhances accuracy and precision in, for example, competitive binding assays resulting in the production of a signal more truly representative of the true concentration of analyte in a sample.
In the assays of this invention, a receptor such as an antibody is generally immobilized in or on a component of the analytical element comprising a material having a net charge that is the same as that of the analyte. The element can be a layer of film; the surface of a cup; a fibrous layer used, for example, in xe2x80x9cdip and readxe2x80x9d format; a bead; a tube surface; or other medium. The receptor is specific for the analyte having the net charge. Either before the receptor is bound to the element or after the receptor is immobilized on an analytical element, sample containing the analyte is added to it. The analyte may have been previously mixed with labeled-analyte. Alternatively, the labeled-analyte could be added later. In any case, analyte and labeled analyte will then compete with each other for sites on the receptor. Because the component of the assay element to which the receptor is bound and/or some other component or surfaces nearby or in contact with the component of the element of the analytical device also has a net charge that is the same as the ligand or labeled ligand, the species which is not meant to be preferentially bound to the receptor will be repelled by the element to which it is affixed. In a subsequent step in which the species not meant to be bound is to be removed (e.g., by washing), it is not able to nonspecifically bind to the element. Accordingly, it is more readily removed from the assay. Thus, in a yet further step in which the bound ligand or bound labeled ligand is exposed to further reagents such as enzyme substrate and then measured for the generation of a signal, there is a reduced level of nonspecifically bound ligand or labeled ligand to generate such an undesirable signal. This results in an analyte measurement that is more accurate and more precise. Alternatively, the label that has been separated from that which is specifically bound to the receptor can be measured with greater accuracy and precision. This assay could also be performed in an inverse format whereby the analyte (ligand) is bound directly or indirectly to the element and the receptor is labeled. Nonspecific binding would still be a problem here, and the addition of a charged polymer would also ameliorate this situation.
Materials useful as the charge bearing substance of this invention include any material to which a receptor can be immobilized and which is capable of carrying a net charge. The substance is not limited to a material that is capable of binding a receptor but it must be capable of bearing a charge. These materials include, for example, metals, ceramics, zeolites, organic polymers, inorganic polymers, oligomers, macromers, ionomers, and semiconductors. Polymers are preferred. Water soluble polymers are more preferred.
The most preferred polymers of this invention are copolymers comprised of cationic vinyl monomers that undergo addition polymerization. For clarity, the term xe2x80x9ccopolymerxe2x80x9d is used to mean any polymer formed by the combination of two or more non-identical monomers and includes species such as terpolymers and the like. Preferably, copolymer compositions used in the invention comprise 20-80% wt (based on total weight) of copolymer of cationic species with the remainder being a diluent comonomer. Cationic vinyl addition monomers comprising quaternary nitrogens having alkyl substituents of one to three carbon atoms have been found suitable when the analyte has a net positive charge as with aminoglycosides. Those having quaternary nitrogen containing groups having carbon, hydrogen, and hetero atoms necessary to complete a substituted or unsubstituted mono or polycylic nitrogen-containing cationic group having about 5-14 ring carbon and hetero atoms have also been found to be particulary suitable in similar circumstances.
In a preferred embodiment, the polymer is made from a monomer that includes a quaternary nitrogen having one or more C1 to C3 alkyl groups, or a monomer having a mono- or poly-cyclic ring between 5 to 14 ring atoms selected from C, S, N, or O, provided at least one of the ring atoms is a quaternary nitrogen.
The following are particularly suitable monomers for the copolymers used to make the analytical elements of this invention. 1-(N,N,N-trimethylammonium)ethyl methacrylate chloride, 2-(N,N,N-trimethylammonium)ethyl acrylate methosulfate, 3-(N,N,N-trimethylammonium-2-hydroxy)propyl methacrylate methosulfate, N-(2-acryloyloxyethyl)-N,N-dimethyl-N-ethylammonium ethosulfate, 2-(N,N,N-trimethylammonium)ethyl methacrylate methosulfate, 3-(N,N,N-trimethylammonium-2-hydroxy)propyl methacrylate chloride, N-(2-acryloyloxyethyl)-N,N-dimethyl-N-ethyl-ammonium chloride, 3-trimethylammonio-1, 1-dimethylpropylacrylamide methosulfate, 3-methyl-1-vinylimidazolium methosulfate, mandp-N-vinylbenzyl-N,N-dimethyl-N-cyclohexylammonium chloride (60:40), 4-vinyl-N-methylpyridinium methosulfate, mandp-N-vinylbenzyl-N,N,N-triethylammonium chloride, mandp-N-vinylbenzyl-N,N,N-trimethylammonium chloride (60:40), mandp-N-vinylbenyl-N-benzyl-N,N-dimethylammonium chloride (60:40), mandp-N-vinylbenzylpyridinium chloride (60:40), 2-(N,N,N-trimethylammonium)ethyl methacrylate chloride, N-(2-acryloyloxyethyl)-N,N,N-triethylammonium ethosulfate, and 3-(N,N,N-trimethylammonium-2-hydroxy)propyl methacrylate chloride.
Preferred monomers are of the formula: 
and a monomer of formula 
wherein X is NH or O; R1 is H or CH3; R2, R3, R4, R5 and R6 are independently H, CH3, CH2CH3, R7 is H, CH3, CH2CH3 or cyclohexyl; Y is CONHCH2CH2CH3, CONHCH(CH3)2, COOCH2CH2OH, or 2-pyrrrolidinone-1-yl; and n is 0, 1 or 2.
Diluent monomers are vinyl addition monomers, typically hydrophilic non-ionic acrylamides or methacrylamides or acrylates or methacrylates. A preferred diluent monomer is acrylamide. Other diluent monomers include but are not limited to N-isopropylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and N-vinyl-2-pyrrolidone.
The most preferred copolymer is poly(acrylamide-co-N-(3-methacrylamidopropyl)-N,N,N-trimethylammonium chloride (50/50) wt ratio.
The copolymers of this invention can be prepared using standard emulsion, solution, or suspension polymerization techniques. Numerous patent and non-patent publications are available to the skilled artisan describing polymerization methods useful for preparing copolymers of this invention, for example: Sorenson et al. in Preparative Methods of Polymer Science, 2nd Ed. Wiley and Sons (1968); Stevens, Polymer Chemistry, An Introduction, Addison Wesley Publishing Co. (1975); U.S. Pat. Nos. 4,581,314 and 4,599,389, and 4,201,840. The entire contents of these publications are incorporated herein by reference. U.S. Pat. No. 4,201,840 for example, teaches the preparation of poly acrylamide-co-N-(3-methacrylamidopropyl)-Nxe2x80x2-(3-chloroprop
ionyl)urea by first preparing a solution of 36 g of acrylamide, 9 g of N-(3-methacrylamidopropyl)-Nxe2x80x2-(3-chloropropionyl)urea, and 225 mg of 2,2xe2x80x2-azobis(2-methylpropionitrile) in 405 ml of dimethylsulfoxide. The solution was flushed with nitrogen for xc2xd hour and heated at 60xc2x0 C. overnight to yield a viscous polymer solution. The polymer was isolated by precipitation from acetone, collected by filtration and dried. The desired polymers of this invention can be made in similar fashion. A specific relative amount of one monomer to another monomer in the copolymer can be obtained by using that specific relative amount of each monomer at the start of the polymerization reaction.
In the dry-film immunoassay elements of the present invention, the cationic polymers may be incorporated as a separate layer between the receptor layer (reaction zone) and the spread layer; in the receptor layer; in the spread layer; or in a gravure layer over the spread layer. The polymer may be applied as gravure layer in multiple passes. A gravure layer, as the term is used in this specification, is a layer disposed on the surface of another layer (such as a spread layer) that does not substantially penetrate the layer on which it is disposed. Applying the polymer as a gravure layer in multiple passes is particularly desirable where the polymer is applied as part of a dilute polymer solution. A preferred method is to incorporate the polymer both in the receptor layer and in the gravure layer. The amount of polymer to be incorporated in the coated element may vary from 0.2-2.5 g/m2, a preferred coverage is 0.8 g/m2 (incorporated into the receptor and gravure layer). Incorporation of dry film analytical elements and their construction is taught, for example, in U.S. Pat. No. 4,258,001, No. 4,357,363, No. 5,714,340 and No. 5,928,886; each of which is incorporated herein by reference. Essentially, a plastic film support comprising a material such as polyethylene terephthalate is coated with various layers by the addition of wet slurries. In the case of the charged polymers of this invention, the wet slurry can be worked at room temperature. It is applied to a thickness of preferably about 0.1 to about 1.0 g/m2 (more preferably about 0.2-0.4 g/m2) (based on dry coverage) and air dried.
When the charged polymer is incorporated into the receptor layer, the microgel polymer used in the receptor layer and the charged polymer are combined (along with the additional ingredients used in such layers) with deionized, distilled water and the pH is adjusted to 7.0. The wet coverage of the receptor melt is 45 g/m2. The melt is coated while at room temperature and is dried at elevated temperature, preferably about 95xc2x0 C.
When the charged polymer is incorporated into the element as a separate layer, the melt is prepared as described above for the receptor layer except that the antibody beads, the micorgel polymer, and the leuco dye are omitted.
The following nonlimiting examples further illustrate the invention.